Most bioherbicides are developed from host-specific plant pathogens. These pathogens attack and suppress the growth and expansion of a target weed but are seldom virulent enough to effectively destroy the target plant. The approach we describe is a novel way to enhance the virulence of plant biocontrol agents without producing potentially dangerous metabolites. The basic background behind this study involves aspects of self-regulation of intermediary metabolism in plants and microbes. This approach does not require recombinant genetics thereby decreasing the time and testing involved with biosafety considerations.
Most modern chemical herbicides inhibit a single biosynthetic enzyme in the target plant. This enzyme inhibition renders the plant incapable of producing metabolites essential for plant growth or defense and eventually leads to death of the plant. Glyphosate, sulfonylureas, imidazolinones, 1, 2, 4-triasol and pyrimidines are classic examples of herbicides that interfere with amino acid biosynthesis. Glyphosate inhibits 5xe2x80x2 enol pyruvyl shikimate 3-phosphate synthase (EPSP), the key enzyme in the shikimic acid pathway (Amrhein, 1986). Another target enzyme, acetolactate synthase (ALS) is a unique herbicide target in that several structurally differing compounds inhibit the enzyme (sulfonylureas, imidazolinones, 1, 2, 4-triasol pyrimidines). The activity of ALS is also inhibited by its own biosynthetic end-products (valine and/or isoleucine) efficiently regulating the balanced production of branched amino acids. Accumulation of a single end-product in a branched biosynthetic pathway may lead to shutdown of the entire pathway. For example, isoleucine inhibits ALS preventing not only biosynthesis of isoleucine but also biosynthesis of valine, leucine, and the essential vitamin, pantothenic acid. Accumulation of both isoleucine and valine has a synergistic effect further reducing the activity of the enzyme.
Feedback inhibition of biosynthetic enzymes in other amino acid pathways is also well documented. In higher plants and bacteria, lysine, threonine, and methionine are synthesized in a branched pathway from aspartate (Bryan, 1980; Umbarger and Davis, 1962). The activity of the first enzyme in this pathway (aspartate kinase) is regulated by the concentrations of lysine and threonine. The activity of the third enzyme in the pathway is regulated by the concentration of methionine (Green and Phillips, 1974). Hence, the exogenous application of one of the end-product amino acids leads to repression of the entire pathway, resulting in depletion of the other two amino acids, and eventual starvation.
Mis-regulation by exogenous end-products has been reported in the plant kingdom. Examples include xe2x80x9cfrenching diseasexe2x80x9d of tobacco where unusual strains of Pseudomonas flourescens, a bacterium in the root zone, produce a significant amount of the essential amino acid isoleucine, which inhibits the plant""s growth (Steinberg, 1950, Steinberg, 1946). Among the earliest symptoms of frenching is chlorosis along the margins of young leaves, which spread gradually across the entire leaf surface. As the leaf develops, only the midrib elongates, thereby producing a distorted narrow leaf. Terminal growth is greatly retarded and apical dominance is lost, resulting in a stunted plant with small, distorted leaves. In severe cases, the axillary buds are stimulated into growth (Steinberg, 1950; Lucas, 1965). Steinberg (1950) reported Bacillus cereus also could cause severe xe2x80x9cfrenchingxe2x80x9d symptoms in tobacco seedlings. Higher populations of this nonpathogenic microorganism were found in the rhizosphere of frenched tobacco than in the rhizosphere of normal tobacco. High concentrations of free isoleucine were detected in the leaves of frenched plants (Steinberg, 1946).
Regulatory mutants that are resistant to feedback inhibition or enzyme repression have been reported in bacteria (Adelberg, 1958; Umbarger and Davis, 1962), in fungi (Maiti, 1988), and in plants (Wu et al, 1994; Relton et al, 1986, Carlson, 1973). The biosynthetic activities of these xe2x80x9cmutantxe2x80x9d enzymes were not sensitive to inhibitory concentrations of end-product resulting in continuous production of pathway intermediates and biosynthetic end-products. In bacteria, the accumulated end-product may be excreted from the cell, restoring the balance of the intracellular amino acid pool. This excretion of amino acids by a plant pathogenic bacterium or fungus could disrupt the delicately balanced production of amino acids in infected host plants further increasing the susceptibility to the pathogen or biological control agent.
In some cases, regulatory mutants can be selected by exposing the organism to high concentrations of a single amino acid and selecting for wild-type growth. An alternative approach is to expose the microorganism to lethal concentrations of a toxic amino acid analog. Amino acid analogs may act as false end-product inhibitors or as false repressors for enzymes involved in biosynthesis of amino acids (Rosenthal, 1982; Umbarger and Davis, 1962). In addition, amino acid analogs may be competitively incorporated into proteins altering or eliminating the protein activity. Strains may show resistance to these analogs by one of two ways: 1) they are incapable of taking up the toxic metabolite; or 2) they are insensitive to the regulatory effects of the toxic analog. The mutants that are insensitive to the regulatory effect of analogs are also resistant to the regulatory effect of the corresponding amino acid and may overproduce and therefore, excrete the amino acids end-products of the specific pathway.